Compositions and methods for diagnosing schizophrenia

ABSTRACT

Methods and compositions for detecting schizophrenia based on modification of the dopamine D 4  receptor by addition of an adenosyl group to methionine #313 via methionine adenosyltransferase are described herein. Individuals with schizophrenia have a deficiency in methionine adenosyltransferase activity and a lower amount of modified dopamine D 4  receptor than normal individuals. Methods for screening therapeutic processes, agents and drugs for use in treatment of schizophrenia are also described.

BACKGROUND

Schizophrenia is a devastating neuropsychiatric disorder which affectsapproximately 1% of the population and results in serious disruption inthe lives of afflicted individuals and their families. Common symptomsinclude delusions, conceptual disorganization and visual or auditoryhallucinations, as well as changes in affective behavior. A number ofscales for the rating of symptoms and methods for ascertaining thediagnosis have been developed, including the DSM classification by theAmerican Psychiatric Association (Diagnostic and Statistical Manual ofMental Disorders (4th edition), pp. 273-316 (1994)), which haveattempted to refine the accuracy of clinical diagnosis. However, it islikely that similar symptoms can result from several underlyingabnormalities, and diagnosis relying solely on clinical symptoms isdifficult and controversial, as well as subjective, time-consuming andcostly.

The cause or causes of schizophrenia remain obscure. A defect indopamine pathways of synaptic neuronal function is a central feature ofthe most widely held etiopathogenic theory (known as the DopamineHypothesis), with recent emphasis on the role of D₄ -type dopaminereceptors (Taubes, Science 265:1034-1035 (1994)). However, studies todate have failed to identify abnormalities in the basic receptorstructure, suggesting that dysfunction may result from an alteration inthe dynamic regulation of receptor activity.

Dopamine receptors are members of a large superfamily of Gprotein-coupled receptors which share a high degree of structuralsimilarity while recognizing a widely divergent array of substanceswhich affect cellular function. Recent advances in the study of thesereceptors, including the development of 3-dimensional structural models(Teeter, et al., J. Med Chem 37:2874-2888 (1994)), have led to theidentification of key locations on the receptors which can modulatetheir function and which therefore may be sites of malfunction inschizophrenia (Samama et al., J. Biol. Chem. 268:4625-4636 (1993)). Onesuch critical location or "hot spot" in the dopamine D₄ receptor is amethionine amino acid residue (Van Tol et al., Nature 350:610-614(1991)).

A number of clinical and metabolic studies have documented alteredmethionine metabolism in individuals with schizophrenia (Kelsoe et al.,J. Neurosci. Res. 8:99-103 (1982), Ismail et al., Biol. Psych.13:649-660 (1978), Sargent et al., Biol. Psych. 32:1078-1090 (1992)).Conversely, administration of S-adenosylmethionine has been shown tohave antidepressant benefits (Kemali et al., Biochemical andPharmacological Roles of Adenosylmethionine and the Central NervousSystem, Pergamon Press, pp. 141-147 (1979)). Despite the evidence forthe importance of an abnormality of methionine metabolism, a cogenthypothesis relating these observations to schizophrenia has not emerged.Until now it has not been possible to integrate findings of alteredmethionine metabolism with the central role of dopamine receptorfunction, an integration which would provide an entirely new approach tothe diagnosis and treatment of schizophrenia. A reliable biochemicalassay would not only provide a more rapid and facile basis fordiagnosis, but would also ease the continuing stigma associated with thedisease by providing a clearly definable chemical cause for a diseasewhich is presently considered a "mental disorder".

SUMMARY OF THE INVENTION

This invention is based upon the discovery that a biochemicalabnormality in methionine metabolism associated with schizophrenia islinked to the clinical manifestations of altered dopamineneurotransmission. This discovery provides novel and empiricalapproaches for the diagnosis and treatment of schizophrenia.

This invention pertains to novel biochemical methods for diagnosingschizophrenia and other neuropsychiatric disorders, includingschizo-affective disorders and dementias. The method of the presentinvention is based on the fact that dopamine receptor function isabnormal in individuals with schizophrenia, and upon the discovery,described herein, that methionine residue #313 (human D₄ receptornumbering) of the dopamine D₄ receptor is abnormally modified inschizophrenic individuals. Methionine residue #313 is normally modifiedby the addition of an adenosyl group to its sulfur atom via the actionof a methionine adenosyltransferase (MAT) enzyme; however, individualswith schizophrenia are deficient in MAT activity and, thus, possess alesser amount of the modified dopamine D₄ receptor. This deficiency isevident in a variety of tissues, including brain tissue and blood cells,particularly white blood cells, and is central to the biochemicaldiagnosis of schizophrenia.

In one embodiment of the present invention, the level of MAT or MATactivity in a tissue sample from an individual to be tested is measuredusing standard methods such as measuring the formation of ³H!S-adenosylmethionine from ³ H!methionine as described in Trolin etal., Eur. Neuropsych 4:469-477 (1994). The measured level is thencompared with the corresponding level from a tissue sample of a normalindividual; a lower level of MAT or MAT activity in the testedindividual compared with the normal individual is indicative ofschizophrenia in the tested individual.

According to another embodiment of the present invention, antibodieswhich specifically recognize either the modified form of the receptor orthe unmodified form of the dopamine D4 receptor are produced by methodsdescribed herein. Such antibodies are also the subject of the presentinvention. The antibodies are used alone or in combination to assess theextent of D₄ receptor modification, and can be used in preparations ofwhite blood cells, white blood cell membranes or platelets or other celltypes which contain the D₄ receptor. The level of binding of antibodieswhich specifically recognize the unmodified receptor region whichincludes methionine #313 will be higher in schizophrenic individualsthan in normal individuals. Reciprocally, the level of binding ofantibodies which specifically recognize the modified receptor will besignificantly lower in schizophrenic individuals than in normalindividuals.

In order to normalize the level of binding to the total amount of D4receptor present in the tissue, the binding of the specific antibodiescan be expressed relative to the binding of an antibody which binds to aregion of the D4 receptor which does not undergo modification; that is,the latter antibody recognizes both the modified and unmodified form ofthe receptor (Ab-BOTH).

In an alternate embodiment of the invention, the determination of theamount of modified and unmodified receptors can be made by isolation ofthe receptor protein itself, followed by chemical analysis of individualamino acids or peptide fragments which contain the site of methioninemodification. For instance, proteolytic enzymes or chemicals could beused to cleave the D₄ receptor into peptide fragments or individualamino acids. The presence of a modified methionine residue could bedetected using spectral analysis, chromatography or other standard meansfor determining the occurrence of a modified amino acid. The occurrenceof modified methionine residues is lower in schizophrenic individualsthan in normal individuals.

Alternatively, modification of an isolated receptor could be assayedusing the receptor as a substrate for the MAT enzyme, and measuring theextent of further adenosylation using, for example, the incorporation ofradioactive adenosine triphosphate. In this case, the receptors fromschizophrenic individuals would show a higher rate of modification thanthose from normal individuals, as they have a lower level ofmodification as a baseline.

As described herein, the methods of the present invention can be used todistinguish between modified and unmodified receptors, particularlymodified and unmodified dopamine D₄ receptors. The present methods canalso be used to determine, by standard procedures, relative levels ofmodified and unmodified receptors. Thus, the compositions and methods ofthe present invention have utility for diagnosis of schizophrenia, aswell as for assessment of progression of the disease and assessment ofthe effectiveness of treatment programs for schizophrenia and otherneuropsychiatric disorders by monitoring levels of modified andunmodified receptors and/or levels of enzyme, or enzyme activityparticularly MAT or MAT activity.

This invention also pertains to novel methods for identifyingtherapeutic processes, agents or drugs for treatment of schizophrenia.Processes, agents and drugs identified by the methods described hereincan increase the amount of MAT enzyme or MAT enzyme activity. Forexample, the process, agent or drug can mimic the effect of methionineadenosyltransferase in adding an adenosyl group to methionine #313.Alternatively, the process, agent or drug can increase the ability ofthe D₄ receptor to become adenosylated, possibly by increasing theability of the receptor to achieve the active conformation from which itcan be adenosylated. This can also be achieved by a partial agonist orantagonist. Alternatively, effective therapeutic processes, agents ordrugs can decrease the deadenosylation of S-adenosylmethionine. Forexample, the process, agent or drug can inhibit the effect of theputative phospholipid methyltransferase, adenosylhomocysteine hydrolaseor methionine synthetase enzymes which convert modified methioninereceptors to unmodified methionine receptors. This can be achieved byintroducing a substrate which competes with the adenosylated methioninefor the action of the putative adenosylase. Preferably this competitivesubstrate is not metabolized to methionine.

This invention also pertains to novel methods for determining theeffectiveness of therapeutic processes and agents for treatment ofschizophrenia. In one embodiment of the invention, the effectiveness ofa therapeutic process or agent can be assessed by making an initialdetermination of the amounts of modified and unmodified D₄ receptors ina tissue sample from an individual to be tested, administering theprocess or agent to be assessed, and making a subsequent determinationof amounts of modified and unmodified D₄ receptors in the tissue samplefrom the individual. The corresponding levels of modified and unmodifiedreceptors can be compared; an increase in the amount of modifiedreceptor and/or a decrease in the amount of unmodified receptorindicates that the process or agent is effective for treatingschizophrenia.

In a particular embodiment, the determination of the amounts of modifiedand unmodified receptors is carried out using two types of antibodies,one of which specifically recognizes the modified form of the receptorand the other of which specifically recognizes the unmodified form ofthe receptor. The results can optionally be normalized to the totalamount of D₄ receptor present by expressing the binding of the specificantibodies relative to the binding of an antibody which recognizes boththe modified and unmodified forms of the receptor (Ab-BOTH).

In an alternate embodiment, the effectiveness of a therapeutic processor agent can be assessed by making an initial determination of the levelof MAT or MAT activity in a tissue sample from an individual to betested, administering the process or agent to be assessed, and making asubsequent determination of the level of MAT or MAT activity in a tissuesample from the individual. An increase in the MAT level or MAT activitylevel indicates that the process or agent is effective in treatingschizophrenia. Thus, the present invention has utility for theidentification of agents, processes and drugs for use in the treatmentof schizophrenia, and such agents, processes and drugs are also thesubject of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a schematic illustration of the major pathways forthe metabolism of free methionine.

FIGS. 2 illustrates the dopamine D₄ receptor structure and shows theamino acid sequence (SEQ ID NO: 1) of the human dopamine D₄ receptor andits proposed seven transmembrane helical elements. The cell membrane isshown as a rectangle with the extracellular surface at the top andintracellular surface at the bottom. Methionine #313 (the "hot spot") isindicated with an arrow.

FIG. 3 illustrates a schematic diagram of antibody-based determinationof receptor modification using Ab-MET Ab-SAM, and Ab-BOTH as examples.

FIG. 4 illustrates the cyclical conversion of the D₄ receptor from itsunmodified methionine form (D₄ (MET)) to its modified (adenosylated)form (D₄ (SAM)), to two additional modified forms (D₄ (AdoHCY) and D₄(HCY)), and finally back to its unmodified form. This cyclic pathwaymimics the metabolism of free methionine shown in FIG. 1. The modifiedforms of the receptor (D₄ (SAM), D₄ (AdoHCY) and D₄ (HCY)) arespontaneously active.

FIGS. 5A and 5B illustrate graphic representations of the effect ofcycloleucine on spontaneous G protein binding of ³⁵ S!GTPγS. FIG. 5Aillustrates the ability of cycloleucine, a methionineadenosyltransferase (MAT) inhibitor, to inhibit spontaneous G proteinbinding of ³⁵ S!GTPγS in Chinese hamster ovary cells which express thecloned dopamine D₄ receptor. FIG. 5B shows the inability of cycloleucineto affect spontaneous ³⁵ S!GTPγS binding in Chinese hamster ovary cellswithout the D₄ receptor.

DETAILED DESCRIPTION OF THE INVENTION

The "Dopamine Hypothesis" is the most widely held biochemicalexplanation for the etiology of schizophrenia and theorizes a defect indopamine pathways of synaptic neuronal function, with recent emphasis onthe role of D₄ -type dopamine receptors. Dopamine receptors are membersof the superfamily of G protein-coupled receptors. All G protein-coupledreceptors share the basic structural motif of seventransmembrane-spanning helices, formed as the single polypeptide chaintraverses the plasma membrane (Figure 2, O'Dowd, J. Neurochem 60:804-816(1993)). In the case of the dopamine receptor, the neurotransmitterdopamine diffuses into the central core of the receptor on the outersurface of the cell where critical amino acid residues provide specificrecognition. The binding of dopamine and its recognition by the receptorcause an alteration in the conformation of the receptor, and this"active" conformation conveys the neurotransmitter signal to GTP-bindingG proteins located on the inner surface of the cell membrane.

Five subtypes of dopamine receptors have been identified, designated asD₁, D₂, D₃, D₄ and D₅. Based upon functional and structuralsimilarities, the D₁ and D₅ receptors form a D₁ -like receptor group,and the D₂, D₃ and D₄ receptors comprise a D₂ -like group. Amongdopamine receptors, the D₁ -like receptors primarily complex with andactivate the G protein G_(s), while the D₂ -like receptors activate theG_(i) and/or G_(o) proteins. The potency of neuroleptic drugs intreating schizophrenia has been found to be closely correlated withtheir antagonism of the D₂ -like receptors (Seeman et al., Proc. Nat.Acad. Sci. USA 72:4376-4380 (1975)), and antagonism of the D₄ receptorsubtype provides a better correlation than do the D₂ or D₃ subtypes,implicating the D₄ receptor as the most likely target of neurolepticdrugs (Seeman and Van Tol, Trends Pharmacol Sci 15:264-270 (1994)).

Generally, a receptor must be occupied by its agonist or partial agonistin order to attain its active conformation and convey theneurotransmitter signal. However, in a phenomenon known as "receptorprecoupling", it is possible for a receptor to maintain the activeconformation even in the absence of agonist occupation, although theextent of this phenomenon appears to be dependent upon prior exposure ofthe cells to the appropriate agonist.

Without wishing to be bound by theory, the binding of the agonistapparently induces a conformational change in the receptor, causing itto become active. In this active state, a modifiable amino acid residue(a "hot spot") on the intracellular portion of the receptor, exactly 18residues (5 helical turns) from the agonist binding site, becomesaccessible to a native enzyme. This enzyme modifies the "hot spot" suchthat the modification (typically a phosphorylation in other precoupledreceptors but adenosylation in the D₄ receptor) prevents the receptorfrom returning to the inactive conformation upon departure of theagonist. In fact, the receptor remains in the active conformation, andcontinues to propagate the neurotransmitter signal, until themodification is removed by subsequent enzyme activity (FIG. 4).

As a result of work described herein, the dopamine D₄ receptor,methionine #313 (human D₄ receptor numbering) has been identified assuch a "hot spot"; that is, methionine #313 is a modifiable amino acidresidue on the intracellular portion of the D₄ receptor, located 18residues (exactly 5 helical turns) below the key residue which isutilized by agonists to induce the active receptor conformation. It hasbeen shown that any modification of residues occupying the same positionas dopamine D₄ receptor methionine #313 in other receptors will causethe receptor to become spontaneously active and exhibit precoupling(Samama et al., J. Biol Chem 268:4625-4636 (1993)).

A number of clinical and metabolic studies have documented alteredmethionine metabolism in individuals with schizophrenia (Kelsoe et al.,J. Neurosci Res 8:99-103 (1982), Ismail et al., Biol Psych 13:649-660(1978), Sargent et al., Biol Psych 32:1078-1090 (1992)). Conversely,administration of S-adenosylmethionine has been shown to haveantidepressant benefits (Kemali et al., Biochemical and PharmacologicalRoles of Adenosylmethionine and the Central Nervous System, PergamonPress, pp. 141-147 (1979)). The major metabolic pathways for freemethionine are illustrated in FIG. 1. The conversion of methionine toS-adenosylmethionine is a key step in methionine metabolism, and amethionine adenosyltransferase (MAT) enzyme participates in thisconversion.

It is believed that the binding of dopamine to the D₄ receptor causes analteration of the receptor conformation to the active conformation. As aresult of this alteration, methionine #313 (human D₄ receptor numbering)becomes accessible to MAT, and MAT adds an adenosyl group to the sulfuratom of the methionine. This modification of methionine #313 by MATprevents the re-configuration of the receptor to the inactive form upondeparture of the agonist. Thus, the dopamine D₄ receptor is capable ofspontaneously maintaining its active conformation, i.e., maintaining itsactive conformation without dopamine occupation (FIG. 4). This activeconformation is maintained until the modification (e.g., the adenosylgroup) is removed by subsequent enzyme activity. A deficiency in MATenzyme activity could account for the observed decrease in methionineutilization in schizophrenics.

A number of other neurotransmitter receptors can also be modified,particularly by phosphorylation, at a site which is equivalent tomethionine #313, and it is possible that abnormal levels of receptormodification are associated with other psychiatric disorders or diseasestates. The methods described herein can be used to determineabnormalities in modification of receptors other than the dopamine D₄receptor.

For instance, the α₂ -adrenergic receptor, M₂ - and M₄ -muscarinicreceptors, D₂ - and D₃ -dopaminergic receptors, μ- and δ-opiatereceptors, SS₂ - and SS₃ -somatostatin receptors and 5-HT_(1B),5-HT_(1D) and 5-HT_(2C) receptors all possess a modifiable amino acidresidue on the intracellular portion of the membrane; exactly 18 aminoacid residues (5 helical turns) separate the modifiable amino acid fromthe amino acid residue which is the agonist binding site on theextracellular portion of the receptor. A modifiable amino acid residuelocated at this position is considered a "hot spot", and modification ofresidues at this "hot spot" position of the receptor cause the receptorto become spontaneously active and exhibit precoupling (Samama et al.,J. Biol Chem 268:4625-4636 (1993)). Usually a modifiable amino acid is aserine residue or a threonine residue which can be modified byphosphorylation; however, as the present invention shows, other residuessuch as methionine can be modified.

Thus, the methods described herein can be used to diagnose and monitorneuropsychiatric disorders, in addition to schizophrenia, which resultfrom abnormal receptor modification. Specifically, after the "hot spot"modifiable amino acid residue in the receptor of interest, and/or theenzyme or enzymes responsible for the modification are identified, thelevels of modified, or unmodified receptor or enzyme activity can bedetermined and compared with normal levels. A difference in the levelsof modified receptor, unmodified receptor and/or enzyme activitycompared with normal levels is indicative of abnormality in receptormodification and resultant neuropsychiatric disorder.

The degree of spontaneous activity of the receptor can be monitored bymeasuring the level of G protein activation, and particularly bymeasuring the binding of ³⁵ S!GTPγS. The effect of cycloleucine, a knownMAT inhibitor, on the binding of ³⁵ S!GTPγS was tested by treatingChinese hamster ovary cells expressing the D₄ receptor with 0.1 mM and 1mM cycloleucine prior to membrane preparation (FIG. 5A). The binding of³⁵ S!GTPγS to the membranes was progressively reduced by 0.1 mM and 1 mMcycloleucine pretreatment to levels significantly lower than theuntreated control membranes. Thus, inhibition of MAT, such as withcycloleucine, results in a decrease in G protein activation, confirmingthat MAT is a key factor in spontaneous activity of the dopamine D₄receptor.

The present invention relates to methods for determining the level ofMAT or MAT activity. One method involves a direct determination of thelevel of MAT enzyme or MAT enzyme activity in a tissue sample. Thisdetermination can be carried out using standard techniques for assayingenzyme presence or activity.

An alternative method involves a determination of the levels of D₄receptor which have been modified to include an adenosyl group onmethionine #313, and those which have not been modified. The modifiedreceptor can be in the form of S-adenosyl methionine (D₄ (SAM)),S-adenosyl homocysteine (D₄ (AdoHCY)) or homocysteine (D₄ (HCY)), since(as shown in FIG. 4) a deficiency in MAT activity results in lowerlevels of all three forms. The determination of modified receptor levelcan be carried out using standard techniques. Methionine modificationserves as an indicator of MAT activity because the modification processrequires MAT activity to proceed. Thus, a high level of modifiedreceptor and correspondingly low level of unmodified receptor correlatesto a high level of MAT activity, while a low level of modification andreciprocal high level of unmodified receptor is consistent with a lowlevel of MAT activity.

The methods described herein are useful in diagnosing schizophrenia andother neuropsychiatric disorders. While one enzyme of interest inschizophrenia is MAT, the enzyme or enzymes of interest in otherneuropsychiatric disorders can be protein kinases (e.g., protein kinaseA or protein kinase C) or protein phosphatases. The particular enzymesassayed will depend upon the particular modification of the "hot spot"residue.

According to the present invention, the level of MAT or MAT activity ina tissue sample from an individual to be tested is measured, and thislevel is compared with the corresponding level of MAT or MAT activity ina tissue sample from a normal individual. If the MAT or MAT activitylevel of the tissue sample from the tested individual is less than thatof the normal tissue sample, the results are indicative ofschizophrenia. If the MAT activity level of the tissue sample from thetested individual is equal to or greater than that of the normal tissuesample, the results are not indicative of schizophrenia.

Similarly, the levels of modified and/or unmodified D₄ receptor in atissue sample from an individual to be tested can be measured, and theselevels can be compared with the corresponding levels in a tissue samplefrom a normal individual. If the level of modified receptor is greaterin the normal tissue sample than in the tested sample, and/or the levelof unmodified receptor is lower in the normal tissue sample than in thetested sample, this is consistent with a reduced level of MAT activityin the tested sample, and indicative of schizophrenia.

Regardless of which of the above approaches are used, the determinationof MAT activity is carried out by testing a tissue sample from anindividual of interest. Any tissues which display theschizophrenia-associated alterations in methionine metabolism aresuitable for use in the present invention. Such tissues include braintissue and red and white blood cells. White blood cells, white bloodcell membranes and platelets are particularly useful in the presentinvention because of their accessibility, allowing the methods of thepresent invention to be carried out on a blood sample from theindividual.

Antibodies designed to specifically recognize either the modified orunmodified form of the receptor are particularly useful for measuringthe level of modified D₄ receptor in a tissue sample from an individual.Such antibodies can be used to determine the level of receptormodification by measuring the amount of binding of antibodies whichspecifically recognize the adenosylated methionine #313 in D₄ (SAM), D₄(AdoHCY) and/or D₄ (HCY); the determination and quantification ofantibody binding can be carried out using standard techniques, such asWestern blotting, ELISA and fluorescence-activated cell scanning (FACS)assays.

According to one embodiment of the invention, peptides corresponding tothe unmodified receptor region of interest, e.g., the dopamine D₄receptor region containing unmodified methionine #313, are synthesizedand conjugated to an immunogenic hapten. Various immunogenic haptens areknown in the art, and those of particular utility include keyhole limpethemocyanin (KLH) and bovine serum albumin (BSA). Antibodies are raisedagainst the conjugate which selectively recognize only receptorscontaining the unmodified methionine (Ab-MET, FIG. 3). Standardtechniques, such as those described in Current Protocols in Immunology(Vol. I, Wiley Interscience (1994)), can be used to construct theantibodies of the present invention.

Peptides corresponding to the modified receptor region of interest,e.g., the dopamine D₄ receptor region containing methionine #313 whichhas been modified by addition of an adenosyl group to its sulfur atom inthe D₄ (SAM), D₄ (AdoHCY) and/or D₄ (HCY) forms, can also be synthesizedand conjugated to an immunogenic hapten. Antibodies are raised againstthese conjugates which recognize only the receptor containing themodified methionine. For instance, antibodies can be raised against D₄(SAM) which recognize only the S-adenosyl methionine (SAM) form of theD₄ receptor (Ab-SAM, FIG. 3). Based upon the metabolism of methionineitself, three distinct modified forms of the D₄ receptor can beidentified, as shown in FIG. 4. Since reduced activity of the initialmodifying enzyme (MAT) is thought to be the primary defect inschizophrenia, quantitation of the percentage of unmodified D₄ receptorsto all modified forms of the receptor is a particularly usefuldiagnostic determination.

An antibody (e.g., Ab-BOTH) which recognizes the D₄ receptor withoutregard to its state of modification (e.g., at an extracellular loop) isuseful for detection of the entire D₄ receptor population and foridentifying those cells which contain D₄ receptors and those which donot.

The synthetic peptide which is conjugated to an immunogenic hapten andused to raise antibodies should be long enough to provide uniquerecognition of the target receptor region to the exclusion of otherreceptors or regions thereof. Thus, the appropriate length of thepeptide will vary depending upon the target receptor region; however,generally the length of the peptide is from about 6 amino acids to about15 amino acids. Additional amino acid residues can also be added to thepeptide to facilitate coupling of the peptide to the hapten or toenhance the immunogenicity of the peptide. Alternatively, the unmodifiedmethionine residue itself can be used to raise an antibody whichspecifically recognizes only this residue.

In order to determine the relative levels or ratio of modified tounmodified receptors in a given sample, and thus determine the existenceof a deficiency or excess in MAT activity, each of the specificantibodies can be coupled to a different detection system. For instance,rabbit Ab-MET could be detected using goat anti-rabbit IgG coupled tohorseradish peroxidase, while mouse Ab-SAM could be detected using goatanti-mouse IgG coupled to alkaline phosphatase. The ratio of peroxidaseto phosphatase activity in a sample under standard conditions providesan estimate of the predominance of the unmodified form of the receptorversus the modified form of the receptor.

The amount of modified or unmodified receptor present in a sample canalso be directly compared with the amount of corresponding modified orunmodified receptor in a sample from a normal individual. The levels ofmodified and unmodified receptors in the normal sample can be determinedconcurrently with the determination in the tested sample, or the normaldetermination can be made in advance and used as a baseline level totalcompare future samples against. A higher level of unmodified receptorand/or a lower level of modified receptor in the tested individualcompared with normal levels is indicative of schizophrenia.

The following Examples are offered for the purpose of illustrating thepresent invention and are not to be construed to limit the scope of thepresent invention:

EXAMPLES Assay for Binding of GTP to G Proteins

Chinese hamster ovary (CHO) cells stably expressing a cloned dopamine D₄receptor were grown as monolayers at 37° (95% O₂ /5%CO₂) in alphaminimum essential medium supplemented with 2.5% fetal calf serum, 2.5%horse serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/mlfungizone and 400μg/ml G418.

Cells were washed twice with phosphate-buffered saline (137 mM NaCl, 2.6mM KCl, 10 mM Na₂ HPO₄, 1.8 mM KH₂ PO₄), harvested with a rubberpoliceman, and pelleted at 4° at 200×g. The pellet was resuspended in 5ml/dish of lysis buffer (5 mM Tris.HCL, pH 7.5, 5 mM EDTA, 5 mM EGTA,0.1 mM phenylmethylsulfonyl fluoride) at 4° and was homogenized with aDounce homogenizer. The cell lysate was then centrifuged at 34,000×g for15 minutes, and the pellet was resuspended in membrane buffer (50 mMTris.HCl, pH 7.5, 0.6 mM EDTA, 5 mM MgCl₂, 0.1 mM phenylmethylsulfonylfluoride) on ice. Aliquots were rapidly frozen in liquid nitrogen andstored at -80° until used.

³⁵ S!GTPγS binding was measured with a modification of the assay used byHilf and Jakobs (Eur J Pharmacol 172:155-163 (1989)). Cell membraneswere thawed and diluted with 10 mM Tris.HCl buffer. Membrane protein(4-8 μg) was mixed with reaction mixture (50 mM Tris.HCl, pH 7.5, 5 mMMgCl₂, unless otherwise specified, 1 mM EDTA, 1 mM dithiothreitol, 100mM NaCl, 2 μM GDP and 2-3 nM ³⁵ S!GTPγS, unless otherwise specified), ina total volume of 100 μl. The incubation was started by addition ofreaction mixture to the membrane suspension and was carried out in atleast triplicate for varying times at 25° . The reaction was terminatedby rapid filtration through GF/C filters under vacuum. The filters werewashed four times (with 4 ml of 50 mM Tris.HCl, pH 7.5, containing 5 mMMgCl₂ and 100 mM NaCl) and then counted in scintillation cocktail.Nonspecific binding was determined in the presence of 10 μM GTPγS andsubtracted from total bound radioactivity.

Production of Antibodies

The amino acid sequence of the D₄ dopaminergic receptor was examined andcompared with other receptors in order to identify the sequence whichcorresponds to the carboxyl terminus of the third intracellular loop.This sequence contained a methionine amino acid residue which can bestructurally modified by methionine adenosyltransferase. For antibodyproduction, a decapeptide containing the modified residue in an internalposition, plus an added cysteine residue at the amino terminus forcoupling purposes, was synthesized and purified by standard solid phasepeptide synthesis techniques. The peptide was coupled (i.e., crosslinkedwith N-SIBA) via its cysteine residue to two different hapten proteins,bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), toenhance its immunogenicity. The extent of conjugation was quantified bymeasuring the depletion of the reactive lysine residues in BSA and KLHusing a TNBS assay. Additionally, the same peptide is prepared exceptthat the modifiable residue is in its adenosylated, adenosylhomocysteineor homocysteine form, and this peptide is coupled to BSA and KLH.

Polyclonal Antibody Production

An initial pre-immune serum sample was obtained from each of tworabbits. The KLH-conjugated peptide was dissolved in water and mixedwith an immunologic adjuvant (e.g., Complete Freund's adjuvant orTITERMAX®) to form an emulsion. An amount of emulsion containing 10-25μg of the peptide was injected near each of the inguinal and axillarylymph nodes (i.e., four injection sites) of two rabbits for eachpeptide. Three weeks later a similar injection was given to boost theimmunologic response, the booster injection was repeated six weeks afterinitial immunization. Blood samples were obtained approximately 10 daysafter each booster and analyzed by ELISA for the presence and titer ofthe desired antibodies, using the BSA-peptide conjugate as the source ofantigen. Additional boosters can also be given at 3 week intervals untila desired titer (e.g., 1:50,000) is obtained.

While the crude polyclonal antiserum can be used for assay purposes, theactive antibodies are preferably purified by standard affinitychromatography techniques to reduce background binding by non-specificantibodies. For this purpose the peptide is crosslinked to a columnchromatography matrix (e.g., sepharose), and the antiserum passedthrough the column, allowing the specific antibodies to be retained bythe column. Subsequently, the desired antibodies can be eluted from thecolumn by washing it with buffer of higher ionic strength and pH.

It is also possible to use affinity column chromatography to deplete aserum sample of undesired antibody. For example, a column formed withthe unmodified peptide could be used on a serum sample containingantibodies against the unmodified peptide to remove any antibodies whosebinding is not strictly dependent upon the presence of the modification.ELISA assay of the eluates is used to monitor the effectiveness ofdepletion.

Monoclonal Antibody Production

For each peptide, 10 mice are immunized with the KLH-peptide conjugatealong with adjuvant following the protocol outlined for polyclonalantibodies. When serum titers have reached the desired level, hybridomasare produced by fusing spleen cells (or other types ofantibody-producing cells) from the immunized mice with murine myelomacells (e.g., the Sp2/O-Ag14 cell line, using standard HAT(Hypoxanthine/Aminopterin/Thymidine) medium selection techniques.HAT-sensitive myeloma cells are grown in culture for 8 days andharvested. From 1 to 5×10⁷ myeloma cells and 1 to 5×10⁶antibody-producing cells are mixed in Dulbecco's Modified Eagle's Mediumin the presence of 30-50% polyethyleneglycol (molecular weight=100 to5000) to promote cell fusion. Viruses can also be used to facilitatefusion. Cells are plated out and allowed to grow for 8 to 10 days in HATmedium. During this time only the hybridoma cells will survive, and suchpositive clones are expanded in culture wills and the supernatant fromthese wells is assayed for the presence of the desired antibody byELISA, using the appropriate peptide as an antigen. Further cloning bydilution can yield a cell population producing a pure monoclonalantibody. Larger quantities of antibody are produced by injecting thesecells into the peritoneal cavity of mice along with a mineral oiladjuvant. The ascites fluid which develops in 2 to 3 weeks contains highlevels of the desired antibody.

Assay Methods for Measuring Receptor Modification

Once an antibody has been produced that will bind to the epitope regionexclusively in its unmodified or modified form, the amount of itsbinding to a standard amount of receptor material defines the status ofthe modifiable residue. For instance, antibody which can bind only tothe epitope region of the D₄ receptor in its non-adenosylated form canbe used to detect the predominance of that form if the amount ofantibody binding can be quantitated relative to the total amount ofreceptor material which is present in a given sample. The total amountof receptor material in a sample is quantitated by the binding of asecond antibody to another region of the receptor which is not subjectto modification, or with an antibody which binds both the modified andunmodified forms of the receptor equally well. This type of antibody canbe considered to be "neutral antibodies" with regard to the modificationstatus of the receptor. Thus, for a particular sample, the ratio of thespecific antibody binding to the neutral antibody binding can be used asan index of the modification status. The binding of the antibodies canbe detected and quantitated by a variety of standard techniques,including Western blots of proteins separated by gel electrophoresis,sandwich ELISA assays and fluorescence-activated cell sorting assays.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims:

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 387 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetGlyAsnArgSerThrAlaAspAlaAspGlyLeuLeuAlaGlyArg                              151015                                                                        GlyProAlaAlaGlyAlaSerAlaGlyAlaSerAlaGlyLeuAlaGly                              202530                                                                        GlnGlyAlaAlaAlaLeuValGlyGlyValLeuLeuIleGlyAlaVal                              354045                                                                        LeuAlaGlyAsnSerLeuValCysValSerValAlaThrGluArgAla                              505560                                                                        LeuGlnThrProThrAsnSerPheIleMetSerLeuAlaAlaAlaAsp                              65707580                                                                      LeuLeuLeuAlaLeuLeuValLeuProLeuPheValTyrSerGluVal                              859095                                                                        GlnGlyGlyAlaTrpLeuLeuSerProArgLeuCysAspAlaLeuMet                              100105110                                                                     AlaMetAspValAlaLeuCysThrAlaSerIlePheAsnLeuCysAla                              115120125                                                                     IleSerValAspArgPheValAlaValAlaValProLeuArgTyrAsn                              130135140                                                                     ArgGlnGlyGlySerArgArgGlnLeuLeuLeuIleGlyAlaThrTrp                              145150155160                                                                  LeuLeuSerAlaAlaValAlaAlaProValLeuCysGlyLeuAsnAsp                              165170175                                                                     ValArgGlyArgAspProAlaValCysArgLeuGluAspArgAspTyr                              180185190                                                                     ValValTyrSerSerValCysSerPhePheLeuProCysProLeuMet                              195200205                                                                     LeuLeuLeuTyrTrpAlaThrPheArgGlyLeuGlnArgTrpGluVal                              210215220                                                                     AlaArgArgAlaLysLeuHisGlyArgAlaProArgArgProSerGly                              225230235240                                                                  ProGlyProProSerProThrProProAlaProArgLeuProGlnAsp                              245250255                                                                     ProCysGlyProAspCysAlaProProAlaProGlyLeuProProAsp                              260265270                                                                     ProCysGlySerAsnCysAlaProProAspAlaValArgAlaAlaAla                              275280285                                                                     LeuProProGlnThrProProGlnThrArgArgArgArgArgAlaLys                              290295300                                                                     IleThrGlyArgGluArgLysAlaMetArgValLeuProValValVal                              305310315320                                                                  GlyAlaPheLeuLeuCysTrpThrProPhePheValValHisIleThr                              325330335                                                                     GlnAlaLeuCysProAlaCysSerValProProArgLeuValSerAla                              340345350                                                                     ValThrTrpLeuGlyTyrValAsnSerAlaLeuAsnProValIleTyr                              355360365                                                                     ThrValPheAsnAlaGluPheArgAsnValPheArgLysAlaLeuArg                              370375380                                                                     AlaCysCys                                                                     385                                                                           __________________________________________________________________________

What is claimed is:
 1. An in vitro method of assessing the effectivenessof a therapeutic process or agent for treating schizophrenia in apatient, comprising the steps of:a) making an initial determination ofthe amount of dopamine D₄ receptors in a tissue sample from the patientwhich have and have not been modified by addition of an adenosyl groupto methionine #313; b) administering to the patient the therapeuticprocess or agent to be assessed; c) making a subsequent determination ina sample of the same tissue type used in step (a) of the amount ofdopamine D₄ receptors in the patient which have and have not beenmodified by addition of an adenosyl group to methionine #313; and d)comparing the corresponding amounts of modified and unmodified dopamineD₄ receptors from steps a) and c), wherein an increase in the amount ofmodified receptors and/or a decrease in the amount of receptors whichhave not been modified subsequent to administration of the therapeuticprocess or agent indicates that the therapeutic process or agent iseffective for treating schizophrenia in the patient.
 2. A methodaccording to claim 1 wherein the determination of the amount of dopamineD₄ receptors which have and have not been modified by addition of anadenosyl group to methionine #313 is carried out using antibodies.
 3. Amethod according to claim 1, wherein steps b), c) and d) are carried outmore than once.
 4. A method of identifying therapeutic processes, agentsand drugs useful for treating schizophrenia, comprising the steps of:a)making an initial determination of the amount of dopamine D₄ receptorswhich have been modified by addition of an adenosyl group to methionine#313 in a cell from a cell line which expresses the modified D₄receptor; b) administering to the cell the therapeutic process, agent ordrug to be tested; c) making a subsequent determination of the amount ofdopamine D₄ receptors which have been modified by addition of anadenosyl group to methionine #313 in the cell; and d) comparing thecorresponding amounts of modified receptors from steps a) and c),wherein an increase in the amount of modified receptors subsequent toadministration of the therapeutic process, agent or drug indicates thatthe therapeutic process, agent or drug is useful for treatingschizophrenia.